**6.2.2 Effect of chitosan on archaeal diversity in UASB treating POME**

Khemkhao et al. (2011) found that all methanogen DGGE bands observed in the control were also detected in UASB with chitosan addition. The observed acetotrophic methanogens were the family *Methanosarcinaceae* and the species *Methanosaeta soehngenii*. The observed hydrogenotrophic methanogens were the order *Methanomicrobiales*, the genus *Methanolinea* sp. and the species *Methanoculleus marisnigri*. On the other hand, some of the acetotrophic methanogens were observed in the UASB with chitosan addition, but not in the control. These acetotrophic methanogens were the order *Methanosarcinales,* the species *Methanosaeta thermophila* and *Methanosaeta harundinacea*.

Methanogenic archaea are oxygen–sensitive anaerobes (Bitton, 1994). They can grow into individual cells, filamentous chains, cubes and/or sarcina. Methanogens are subdivided into two subcategories: (i) hydrogenotrophic methanogens and (ii) acetotrophic methanogens.

The mechanism of anaerobic digestion in methane production consists of a series of complex metabolic interactions between various types of microorganisms in the absence of oxygen. Anaerobic digestion is mediated through processes of hydrolysis, acidogenesis, acetogenesis and methanogenesis. Khemkhao et al. (2011) used 16S rRNA targeted denaturing gradient gel electrophoresis (DGGE) fingerprints to study the microbial communities during anaerobic digestion. They found that bacteria and methanogens could both be detected in

In their experiments, Khemkhao et al. (2011) found that DGGE patterns of bacterial diversity of the three bacterial groups, hydrolytic, acidogenic and acetogenic, persisted at all operating temperatures. However, the distribution of their members among bacteria in each group did show small changes under the different operating conditions. By the end of the operating period, the UASB with chitosan addition was found to contain a lower proportion of hydrolytic bacteria and a higher proportion of acidogenic bacteria than the control. However, the diversity of acetogenic bacteria was found to be similar in the two reactors.

Sulfate-reducing bacteria were detected in the control but not in the chitosan reactor.

bacteria) convert the fatty acids, alcohols and ketones into acetate, CO2 and H2.

**6.2.2 Effect of chitosan on archaeal diversity in UASB treating POME** 

*thermophila* and *Methanosaeta harundinacea*.

methanogens.

It is known (Bitton, 1994) that hydrolytic, acidogenic and acetogenic bacteria work together to degrade complex organic matters into acetate, CO2 and H2. Hydrolytic bacteria begin the process of degradation by breaking down complex organic molecules such as proteins, cellulose, lignin and lipids into soluble monomer molecules by extracellular enzymes, i.e., proteases, cellulases and lipases. The monomer molecules produced are amino acids, glucose, fatty acids and glycerol. These monomers are then degraded by the acidogenic (acid-forming) group of bacteria which convert them into organic acids, alcohols and ketones, acetate, CO2, and H2. The organic acids produced include acetic, propionic, formic, lactic, butyric, and succinic acids. The alcohols and ketones produced are ethanol, methanol, glycerol and acetone. In the final stage, the acetogenic bacteria (acetate and H2-producing

Khemkhao et al. (2011) found that all methanogen DGGE bands observed in the control were also detected in UASB with chitosan addition. The observed acetotrophic methanogens were the family *Methanosarcinaceae* and the species *Methanosaeta soehngenii*. The observed hydrogenotrophic methanogens were the order *Methanomicrobiales*, the genus *Methanolinea* sp. and the species *Methanoculleus marisnigri*. On the other hand, some of the acetotrophic methanogens were observed in the UASB with chitosan addition, but not in the control. These acetotrophic methanogens were the order *Methanosarcinales,* the species *Methanosaeta* 

Methanogenic archaea are oxygen–sensitive anaerobes (Bitton, 1994). They can grow into individual cells, filamentous chains, cubes and/or sarcina. Methanogens are subdivided into two subcategories: (i) hydrogenotrophic methanogens and (ii) acetotrophic

**6.2 Effect of chitosan on microbial diversity in UASB treating POME** 

the UASB reactors operating both with and without chitosan addition.

**6.2.1 Effect of chitosan on bacterial diversity in UASB treating POME** 

Hydrogenotrophic methanogens convert H2 and CO2 into CH4. Acetotrophic methanogens convert acetate into CH4 and CO2. The acetotrophic methanogens grow slower than the acid-forming bacteria. About two-thirds of CH4 is derived from acetate conversion by acetotrophic methanogens. The other third is the result of H2 and CO2 reduction by hydrogenotrophic methanogens.

As stated above (Khemkhao et al., 2011), lower biomass washout was observed from the UASB with chitosan addition than from the control, especially at higher biogas production rates. The DGGE analysis shows that UASB with chitosan addition contains higher populations of *Methanosaeta* species than the control. It can be concluded that the chitosan helped to retain these methanogens, thus resulting in higher populations of acetotrophic methanogens.

Tiwari et al. (2005) and Tiwari et al. (2006) have reported that acetotrophic methanogens significantly accelerate granule development. Higher population of acetotrophic methanogens may in turn lead to higher methane production in the reactors with chitosan addition.

Chitosan has been reported to act like an ECP in enhancing the aggregation of acidogens. As shown in Fig. 7, the aggregated acidogens then form granules with highly elastic outer

Fig. 7. Scheme of granule formation. Top: Surface tension model according to Thaveesri et al. (1995) and Hulshoff Pol et al. (2004). Middle: Some circumstances in the control reactor. Bottom: Enhanced aggregation by chitosan in UASB with chitosan addition (from Khemkhao et al., 2011)

Enhancing Biogas Production and UASB Start-Up by Chitosan Addition 339

For the same amount of chitosan, chitosan in the solution form was shown to be significantly better at enhancing the granulation process and the UASB performance than

For POME treatment, the biogas production rate and the COD removal of the UASB with chitosan addition was on an average 16% and 5%, respectively, higher than that of the control. A DGGE analysis indicates that the chitosan helped to retain the methanogens in the genus *Methanosaeta*, thus resulting in higher populations of acetotrophic methanogens. Further investigations are required to determine optimal chitosan dosages and the optimal times to add chitosan under thermophilic conditions (Khemkhao et al.,

The authors are grateful to the King Prajadhipok and Queen Rambhai Barni Memorial Foundation for financial support to S. Lertsittichai, to Thailand Research Fund (TRF-Master Research Grant, Grant No. MRG-OSMEP505E225) for the financial support to B. Nuntakumjorn and to Thailand Graduate Institute of Science and Technology (TGIST) and the Joint Graduate School of Energy and Environment (JGSEE) for the financial supports to M. Khemkhao. We also would like to acknowledge Faculty of Engineering, King Mongkut's University of Technology North Bangkok for supporting the publication fee. The authors would like to thank Ngaung-Khaem water quality control plant for providing sludge, Suksomboon Palm Oil Co., Ltd. for wastewater samples and Taming Enterprises Co., Ltd. for providing chitosan samples. Special thanks to Dr. Elvin Moore for his critical reading of

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hydrophilic layers around a core of methanogens. According to Hulshoff Pol et al. (2004) and Thaveesri et al. (1995), the acidogens (round and rod cells) aggregate by forming ECP. Dispersed cells are washed out, while some methanogens (rectangular cells) are enclosed inside, becoming the nucleus of a granule with an outer elastic hydrophilic layer formed by ECP-rich acidogens and an inner core of hydrophobic methanogens. Chitosan has been thought to act like ECP in aggregating anaerobic sludge (El-Mamouni et al., 1998). Therefore it may increase the elasticity of outer hydrophilic layers of the granular samples. In UASB with chitosan addition, the growing methanogens are better protected inside an acidogenic layer and may become less susceptible to adhesion to gas bubbles (filled circles) and consequently may be less washed out from the reactor than those in the control.

The polymer additives appear to play a similar role to naturally secreted ECP in aggregating anaerobic sludge. The addition of polymers to anaerobic systems changes the surface properties of bacteria to promote association of individual cells. Polymer may form a solid and stable three-dimensional matrix within which bacteria multiply and daughter cells are then confined (Liu et al., 2002; Show et al., 2006a; Uyanik et al., 2002).

In addition, Show et al. (2006b) have reported that adding an appropriate dosage of polymer in the seeding stage accelerates the start-up time by approximately 50% and the granule formation by approximately 30%. In addition, granules developed in polymer-assisted reactors exhibited better settleability, strength and methanogenic activity at all OLRs tested. Positively charged polymer forms bridges among the negatively charged bacterial cells through electrostatic charge attraction. The bridging effect would enable greater interaction between biosolids resulting in preferential development and enhancement of biogranulation in UASB reactors (Show et al., 2006a).

In the experiments of Khemkhao et al. (2011), the UASB reactor with chitosan addition was treated with a one-time chitosan dose of 2 mg chitosan/g VSS on the first operating day. The performance of the UASB reactor may be further enhanced by more injections of the chitosan solution. However, the evidence from the one-time chitosan dose of 2 mg chitosan/g VSS on the first operating day was that the initial stage of granulation was very important for forming high quality granules.
